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Fractionation profiling: a fast and versatile approach for mapping vesicle proteomes and protein-protein interactions.

Borner GH, Hein MY, Hirst J, Edgar JR, Mann M, Robinson MS - Mol. Biol. Cell (2014)

Bottom Line: Functionally associated groups of proteins are revealed through cluster analysis.Of importance, the cluster analysis extends to all profiled proteins and thus identifies a diverse range of known and novel cytosolic and membrane-associated protein complexes.In addition, it provides a versatile tool for the rapid generation of large-scale protein interaction maps.

View Article: PubMed Central - PubMed

Affiliation: Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany borner@biochem.mpg.de.

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Fractionation profiling predicts novel protein complexes and trafficking pathways in HeLa cells. (A–F) As a primer for a systematic analysis of novel protein–protein associations, the Predictor database was searched for pairs of near-identical profiles. We identified 989 such pairs, derived from 266 proteins in 58 groups (Supplemental Table S5). Clustering by PCA (Supplemental Figure S2) revealed many known stable protein complexes but also novel associations among uncharacterized proteins. A few examples are shown here. (A, B) The SNX4/SNX30 dimer. (A) Fractionation profiles, showing the protein abundance in each fraction relative to the reference (log2 scale). (B) Estimated stoichiometry. (C, D) The C17orf75/WDR11/FAM91A trimer. A previous report suggested the association of C17orf75 and WDR11; profiling clearly indicates a stable complex that includes FAM91A. (C) Profiles. (D) Estimated stoichiometry. (E, F) The LOH12CR1/C10orf32 dimer. (E) Profiles. (F) Estimated stoichiometry. (G, H) Cargo of the ARF6-dependent endocytic pathway. CD44, CD98 (SLC3A2 heavy chain, SLC7A5 light chain), ICAM1, and BSG (CD147) are known cargo proteins of a clathrin-independent endocytic pathway. (G) Profiles of these proteins are almost identical, confirming their similar trafficking itineraries. A further protein, SLC16A1 (monocarboxylate transporter 1), is identified as part of the same cluster. (H) PCA of individual profiles shows that the ARF6-cargo cluster (red) is distinct from flotillins (blue) and caveolins (green). SLC16A1 (black) is part of the ARF6 cluster. Error bars of abundance scores correspond to MADs (n = 6 replicate measurements).
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Figure 5: Fractionation profiling predicts novel protein complexes and trafficking pathways in HeLa cells. (A–F) As a primer for a systematic analysis of novel protein–protein associations, the Predictor database was searched for pairs of near-identical profiles. We identified 989 such pairs, derived from 266 proteins in 58 groups (Supplemental Table S5). Clustering by PCA (Supplemental Figure S2) revealed many known stable protein complexes but also novel associations among uncharacterized proteins. A few examples are shown here. (A, B) The SNX4/SNX30 dimer. (A) Fractionation profiles, showing the protein abundance in each fraction relative to the reference (log2 scale). (B) Estimated stoichiometry. (C, D) The C17orf75/WDR11/FAM91A trimer. A previous report suggested the association of C17orf75 and WDR11; profiling clearly indicates a stable complex that includes FAM91A. (C) Profiles. (D) Estimated stoichiometry. (E, F) The LOH12CR1/C10orf32 dimer. (E) Profiles. (F) Estimated stoichiometry. (G, H) Cargo of the ARF6-dependent endocytic pathway. CD44, CD98 (SLC3A2 heavy chain, SLC7A5 light chain), ICAM1, and BSG (CD147) are known cargo proteins of a clathrin-independent endocytic pathway. (G) Profiles of these proteins are almost identical, confirming their similar trafficking itineraries. A further protein, SLC16A1 (monocarboxylate transporter 1), is identified as part of the same cluster. (H) PCA of individual profiles shows that the ARF6-cargo cluster (red) is distinct from flotillins (blue) and caveolins (green). SLC16A1 (black) is part of the ARF6 cluster. Error bars of abundance scores correspond to MADs (n = 6 replicate measurements).

Mentions: The foregoing data demonstrate the predictive power of our approach for known protein complexes. However, profiling also predicts the existence of novel complexes (Figure 5, Supplemental Figure S2, and Supplemental Table S5). For example, we provide the first evidence that the BAR-domain proteins SNX4 and SNX30 (van Weering et al., 2010) form a stable dimer (Figure 5, A and B). A recent study identified a novel protein dimer C17orf75/WDR11, implicated in protection against ricin toxicity (Bassik et al., 2013). Our profiling data suggest that these proteins are in fact part of a trimeric complex, which also includes the protein FAM91A1 (Figure 5, C and D). The protein C10orf32 has been implicated in susceptibility to arsenic poisoning (Pierce et al., 2012); here we propose that C10orf32 is in complex with the uncharacterized protein LOIH12CR1 (Figure 5, E and F). These examples illustrate that our approach covers a broad spectrum of important novel protein associations.


Fractionation profiling: a fast and versatile approach for mapping vesicle proteomes and protein-protein interactions.

Borner GH, Hein MY, Hirst J, Edgar JR, Mann M, Robinson MS - Mol. Biol. Cell (2014)

Fractionation profiling predicts novel protein complexes and trafficking pathways in HeLa cells. (A–F) As a primer for a systematic analysis of novel protein–protein associations, the Predictor database was searched for pairs of near-identical profiles. We identified 989 such pairs, derived from 266 proteins in 58 groups (Supplemental Table S5). Clustering by PCA (Supplemental Figure S2) revealed many known stable protein complexes but also novel associations among uncharacterized proteins. A few examples are shown here. (A, B) The SNX4/SNX30 dimer. (A) Fractionation profiles, showing the protein abundance in each fraction relative to the reference (log2 scale). (B) Estimated stoichiometry. (C, D) The C17orf75/WDR11/FAM91A trimer. A previous report suggested the association of C17orf75 and WDR11; profiling clearly indicates a stable complex that includes FAM91A. (C) Profiles. (D) Estimated stoichiometry. (E, F) The LOH12CR1/C10orf32 dimer. (E) Profiles. (F) Estimated stoichiometry. (G, H) Cargo of the ARF6-dependent endocytic pathway. CD44, CD98 (SLC3A2 heavy chain, SLC7A5 light chain), ICAM1, and BSG (CD147) are known cargo proteins of a clathrin-independent endocytic pathway. (G) Profiles of these proteins are almost identical, confirming their similar trafficking itineraries. A further protein, SLC16A1 (monocarboxylate transporter 1), is identified as part of the same cluster. (H) PCA of individual profiles shows that the ARF6-cargo cluster (red) is distinct from flotillins (blue) and caveolins (green). SLC16A1 (black) is part of the ARF6 cluster. Error bars of abundance scores correspond to MADs (n = 6 replicate measurements).
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Related In: Results  -  Collection

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Figure 5: Fractionation profiling predicts novel protein complexes and trafficking pathways in HeLa cells. (A–F) As a primer for a systematic analysis of novel protein–protein associations, the Predictor database was searched for pairs of near-identical profiles. We identified 989 such pairs, derived from 266 proteins in 58 groups (Supplemental Table S5). Clustering by PCA (Supplemental Figure S2) revealed many known stable protein complexes but also novel associations among uncharacterized proteins. A few examples are shown here. (A, B) The SNX4/SNX30 dimer. (A) Fractionation profiles, showing the protein abundance in each fraction relative to the reference (log2 scale). (B) Estimated stoichiometry. (C, D) The C17orf75/WDR11/FAM91A trimer. A previous report suggested the association of C17orf75 and WDR11; profiling clearly indicates a stable complex that includes FAM91A. (C) Profiles. (D) Estimated stoichiometry. (E, F) The LOH12CR1/C10orf32 dimer. (E) Profiles. (F) Estimated stoichiometry. (G, H) Cargo of the ARF6-dependent endocytic pathway. CD44, CD98 (SLC3A2 heavy chain, SLC7A5 light chain), ICAM1, and BSG (CD147) are known cargo proteins of a clathrin-independent endocytic pathway. (G) Profiles of these proteins are almost identical, confirming their similar trafficking itineraries. A further protein, SLC16A1 (monocarboxylate transporter 1), is identified as part of the same cluster. (H) PCA of individual profiles shows that the ARF6-cargo cluster (red) is distinct from flotillins (blue) and caveolins (green). SLC16A1 (black) is part of the ARF6 cluster. Error bars of abundance scores correspond to MADs (n = 6 replicate measurements).
Mentions: The foregoing data demonstrate the predictive power of our approach for known protein complexes. However, profiling also predicts the existence of novel complexes (Figure 5, Supplemental Figure S2, and Supplemental Table S5). For example, we provide the first evidence that the BAR-domain proteins SNX4 and SNX30 (van Weering et al., 2010) form a stable dimer (Figure 5, A and B). A recent study identified a novel protein dimer C17orf75/WDR11, implicated in protection against ricin toxicity (Bassik et al., 2013). Our profiling data suggest that these proteins are in fact part of a trimeric complex, which also includes the protein FAM91A1 (Figure 5, C and D). The protein C10orf32 has been implicated in susceptibility to arsenic poisoning (Pierce et al., 2012); here we propose that C10orf32 is in complex with the uncharacterized protein LOIH12CR1 (Figure 5, E and F). These examples illustrate that our approach covers a broad spectrum of important novel protein associations.

Bottom Line: Functionally associated groups of proteins are revealed through cluster analysis.Of importance, the cluster analysis extends to all profiled proteins and thus identifies a diverse range of known and novel cytosolic and membrane-associated protein complexes.In addition, it provides a versatile tool for the rapid generation of large-scale protein interaction maps.

View Article: PubMed Central - PubMed

Affiliation: Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany borner@biochem.mpg.de.

Show MeSH
Related in: MedlinePlus